In the measurement of fluorescence and exitation spectra it is customary to illuminate a sample with monochromatic light from an intense source and to observe the fluorescence emitted by the sample with a monochromator and a photoelectric detection system.
Conventional fluorescence detectors are based on monochromators and an incoherent light source that have essentially the same geometry. Both excitation and emission monochromators lie in the same plane. The cuvette or flow cell is illuminated with excitation light on one side and fluorescence is collected at right angles. The slits of the excitation and emission monochromators are aligned with the long axis of the cell that is perpendicular to the plane of the optics. The cross-section of the cell of conventional detectors in the plane of the optics is typically square.
Standard detection flow cells used in conventional liquid chromatography instruments have the disadvantage that their cell pathways, as a function of their design, are unfortunately short. The width of the emission face of the cell is mapped by collection optics onto the width of the emission monochromator entrance slit. The desired spectral resolution sets a limit to the width of the emission slits, and therefore to the width of the flow cell or cuvette. An exciting beam of light is transmitted through the flow cell to cause fluorescent emission. The amount or intensity of the fluorescent emission is in direct relationship with the path-length of the exciting beam within the sample. As sample volumes are reduced the resulting path-length is proportionally decreased causing diminished sensitivity of the detector.
Fluorescence measuring apparatus of the foregoing type exhibit certain disadvantages. One of the more significant problems is the comparatively low magnitude of the output signal due to the limits encountered by the relatively short path-length. In the usual form of apparatus, light from an intensely bright light source such as a high pressure Xenon arc is focused on the entrance slit of the excitation monochromator, and an image of the exit slit is focused on a sample by means of a first optical system. Fluorescence from the sample is collected by a second optical system and focused on the entrance slit of an emission monochromator such that the signal at the exit slit of the emission monchromator is directed to a detector. A sensitive detector such as a photomultiplier is placed after the emission monochromator exit slit to measure the fluorescent signal at the selected wavelength. Unfortunately, this conventional method of fluorescent sample detection is degraded as the size of the sample is reduced.
In light of this significant detection limitation, caused by a short excitation path-length, there have been a number of attempts employed in the prior art to increase excitation path-lengths, and hence the sensitivity of the analysis.
Work has been done in Raman and fluorescence detection using light guiding flow cells with axial illumination in order to increase the absorbing path-length. In this approach lasers are used for excitation and emission that is collected axially, either in the forward or back-scattered direction. These cells are made from capillary tubing where the analyte solution refractive index exceeds that of the tube walls and the excitation light is guided by total internal reflection. However, in spite of their apparent demonstrated advantages, they currently receive little use because silica-based glasses have high refractive indices (n>1.46). The requirement for total internal reflection dictates that the refractive index of the core sample liquid exceed that of the capillary wall, which severely limits utility.
Hollow glass or silica waveguides used with low-refractive index liquids have drawbacks from an optical point of view. Since they function through internal reflection at the external surface of the glass tube they allow light to propagate through the tubing wall as well as the liquid core. From a spectroscopic point of view, propagation in the glass wall has several adverse consequences. Propagation through the glass reduces the path-length of light in the liquid. Also, particularly in Raman studies, propagation in the glass wall results in the generation of silica bands, contributing to unwanted background noise.
The substitution of Telfon AF tubing with a refractive index less than water overcomes some of these difficulties. In these light guiding cells, excitation path length is increased. However, the emitted fluorescence must travel the length of the light guiding section of the sample liquid and may be subject to re-absorption unless the solutions are extremely dilute. Self absorption can severely limit the linear dynamic range of sample concentration in a light guiding cell.